Amplification of nucleic acids by polymerase chain reaction (PCR) has many applications in biomedical research, diagnostics and biotechnology. The unique specificity of PCR enables selective amplification of a particular nucleic acid sequence in the presence of overwhelming amount of other sequences. Furthermore, PCR can distinguish a target sequence from another sequence that is different by as little as a single base-pair. For example, allele-specific PCR (AS-PCR) is capable of detecting small alterations in DNA and even single nucleotide mutations in the presence of the wild-type, non-mutant DNA (U.S. Pat. No. 6,627,402). In an allele-specific PCR assay, at least one primer is allele-specific, i.e. designed to preferentially match the target sequence (a specific variant of the sequence), but contains discriminating mismatches with non-target sequences (other variants of the sequence). Ideally, primer extension occurs only when the allele-specific primer is hybridized to the target sequence. In a successful allele-specific PCR, the target variant of the nucleic acid is amplified, while the other non-target variants are not, at least not to a detectable level. Unfortunately, with many targets, this ideal is not achievable. It is common that in later cycles of PCR, amplification of the non-target variants of the sequence also becomes detectable. This phenomenon is called “breakthrough amplification.” Even though the AS-PCR primers are perfectly complementary (or at least, share the greater degree of complementarity) with the target sequence and are mismatched (or have more mismatches) with non-target sequences, often amplification of the non-target sequences cannot be completely avoided.
Breakthrough amplification is of special concern in assays where the sample contains small amounts of the target sequence and large amounts of the non-target sequence. For example, in an assay targeting a somatic mutation in a tumor, only a fraction of cells from the patient's sample are tumor cells. A fraction of tumor cells may contain mutations indicating susceptibility to a particular anti-tumor drug (mutations described in U.S. Pat. Nos. 7,294,468 and 7,960,118). In such a sample, a small number of the target (mutant) sequences are mixed with a large number of non-target (non-mutant) sequences. Breakthrough amplification of the non-mutant sequence would produce a false-positive result, falsely indicating the presence of a mutation and misdirecting the patient's therapy. If the specificity of the assay is limited by the breakthrough amplification, so is the clinical utility of the assay.
Various means of preventing or reducing non-specific amplification have been proposed (for example, chemical modifications that affect the specificity of amplification primers, see U.S. Pat. No. 6,011,611; using a blocker oligonucleotide, see U.S. Application Pub. No. 200953720). However, these methods are not always successful in entirely eliminating the breakthrough amplification. Accordingly, there is a need for alternative methods of preventing or minimizing breakthrough amplification in a nucleic acid amplification reaction.